Extracellular matrix vesicles are believed to play a key role in the initiation of calcification in cartilage, bone and dentin, but their mechanism of action is not fully understood. The objective of the proposed research is to elucidate the mechanism of endochondral calcification with special emphasis on the role of matrix vesicles. Attention will be focused on 1) determining factors involved in the formation and release of matrix vesicles by cells, 2) continued characterization of their enzymes, proteins, mineral forms and membrane constituents, and 3) further study of the metabolism of Ca and Pi by matrix vesicles during the induction of mineral formation. Studies will be aimed at a) elucidating the role of local nutritional factors on energy metabolism and its effect on cellular Ca metabolism, b) determining the effect of cellular Ca metabolism on the formation of calcifiable matrix vesicles, c) further elucidating the role of alkaline phosphatase and associated proteins in the induction of mineral formation by matrix vesicles. We will continue to use epiphyseal growth plate cartilage from long bones of rapidly growing chickens as the source of our tissue and cells. This source provides abundant, actively calcifying material for isolation of cells and matrix vesicles, and for studying their metabolism. Techniques used will be tissue culture, cell fractionation, 45Ca- and 32Pi-metabolism, chemical and physical methods: chromatography (column, thin-layer, HPLC, 2-dimensional, etc.) electrophoresis (PAGE, SDS-PAGE, isoelectric focusing), enzymology (purification, kinetic analyses), spectroscopy (UV, IR, NMR, MAS-NMR), electron microscopy (transmission, scanning, freeze-fracture), x-ray diffraction, etc.